Regulation of protein secretion is a key homeostatic mechanism in both the nervous and endocrine systems. Many secretagogues are known to act by increasing intracellular cyclic AMP (cAMP) levels and activating a cAMP-dependent protein kinase. Recent molecular biological experiments have shown a multiplicity of isoforms for the regulatory (R) and catalytic (C) subunits for cAMP-dependent protein kinase although the functional relevance of these isoforms is unknown. Proopiomelanocortin (POMC) is the precursor to both beta-endorphin and adrenocorticotrophic hormone (ACTH) and the regulation of secretion of POMC-derived peptide has been well studied. An anterior pituitary tumor cell line, designated AtT-20, has been characterized that mimics the cAMP-mediated regulation of POMC-derived peptide secretion by corticotropin releasing factor, epinephrine, and somatostatin seen in normal anterior pituitary corticotrophs. This research proposal describes biochemical, immunological, and molecular biological experiments using the AtT-20 cell line which are designed to determine the functional roles of the R and C subunit isoforms in the cAMP regulation of POMC-derived peptide secretion. The specific aims include: (1) characterization of the R and C subunit isoforms present in AtT-20 cells, (2) identification of which subunit isoforms are involved in mediating the cAMP response to various secretagogues by identifying the components of activated holoenzyme complexes and by overexpressing mRNAs for the various subunit isoforms, (3) determining the structural features of the C subunits that are necessary for mediating the cAMP regulation of protein secretion, and (4) an initial characterization of substrate proteins for the C subunit in AtT-20 cells. In the short term, these experiments are expected to increase our understanding of the cAMP-dependent protein kinase as a signal transducing system and in the long term the knowledge derived from these experiments are expected to facilitate identification of important physiological substrate proteins for the cAMP- dependent protein kinase involved in the regulation of protein secretion.